克隆、原核表达和鉴定牛重组SHH蛋白
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摘要
Sonic Hedgehog(SHH)是Hedgehog家族的一员,其不仅参与胚胎发育的基本过程,同时也参与脂肪生成和肌肉生成。SHH在动物肌肉和脂肪沉积上的双向作用提示它是调控肌内脂肪含量(IMF)的重要候选基因。本研究中,使用重叠PCR法克隆了去除信号肽序列的牛SHH(btSHH)基因,然后在大肠杆菌BL21中诱导表达了btSHH蛋白。使用镍亲和层析试剂盒纯化了原核表达的btSHH,并用Western blot验证了目标蛋白的正确性。将纯化后的btSHH添加到前脂肪细胞3T3-L1的培养基中,并诱导细胞分化后表明,重组btSHH蛋白能够显著地抑制脂肪沉积过程。本研究结果为未来肉牛生产中应用btSHH提供理论基础。
Sonic Hedgehog(SHH), a member of the Hedgehog family, not only participates in the fundamental processes of embryonic development, but is also involved in adipogenesis and myogenesis. The dual effects SHH on the development of adipose and muscle tissues indicate that it might be a candidate regulatory factor for intramuscular fat(IMF) deposition in farm animals. In the present study, the Bos taurus SHH(btSHH) gene without the signal peptide sequence was cloned by overlap PCR, and then the btSHH protein was induced to express on E. coli BL21 and was purified by Ni-affinity chromatography. The purified btSHH was verified by western blot and then added to the culture medium of pre-adipocyte 3T3-L1 during the induced differentiation process. The Oil Red O staining showed that the recombinant btSHH could significantly inhibit the adipogenesis. This research might provide a basis for the application of btSHH in beef cattle productions in the future.
Sonic Hedgehog(SHH), a member of the Hedgehog family, not only participates in the fundamental processes of embryonic development, but is also involved in adipogenesis and myogenesis. The dual effects SHH on the development of adipose and muscle tissues indicate that it might be a candidate regulatory factor for intramuscular fat(IMF) deposition in farm animals. In the present study, the Bos taurus SHH(btSHH) gene without the signal peptide sequence was cloned by overlap PCR, and then the btSHH protein was induced to express on E. coli BL21 and was purified by Ni-affinity chromatography. The purified btSHH was verified by western blot and then added to the culture medium of pre-adipocyte 3T3-L1 during the induced differentiation process. The Oil Red O staining showed that the recombinant btSHH could significantly inhibit the adipogenesis. This research might provide a basis for the application of btSHH in beef cattle productions in the future.
引文
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[2] Cohen M M Jr.The hedgehog signaling network[J].Am J Med Genet A,2003,123A(1):5-28.
[3] Varjosalo M,Taipale J.Hedgehog signaling[J].J Cell Sci,2007,120(Pt1):3-6.
[4] Pospisilik J A,Schramek D,Schnidar H,et al.Drosophila genome-wide obesity screen reveals hedgehog as a determinant of brown versus white adipose cell fate[J].Cell,2010,140(1):148-160.
[5] Voronova A,Coyne E,Al Madhoun A,et al.Hedgehog signaling regulates MyoD expression and activity[J].J Biol Chem,2013,288(6):4389-4404.
[6] Borycki A G,Brunk B,Tajbakhsh S,et al.Sonic hedgehog controls epaxial muscle determination through Myf5 activation[J].Development,1999,126(18):4053-4063.
[7] Straface G,Aprahamian T,Flex A,et al.Sonic hedgehog regulates angiogenesis and myogenesis during post-natal skeletal muscle regeneration[J].J Cell Mol Med,2009,13(8B):2424-2435.
[8] Wood J D,Enser M,Fisher A V,et al.Fat deposition,fatty acid composition and meat quality:A review[J].Meat Sci,2008,78(4):343-358.
[9] Webb E C,O’Neill H A.The animal fat paradox and meat quality[J].Meat Sci,2008,80(1):28-36.
[10] Emanuelsson O,Brunak S,von Heijne G,et al.Locating proteins in the cell using TargetP,SignalP and related tools[J].Nat Protocols,2007,2(4):953-971.
[11] James A W,Leucht P,Levi B,et al.Sonic Hedgehog influences the balance of osteogenesis and adipogenesis in mouse adipose-derived stromal cells[J].Tissue Eng Part A,2010,16(8):2605-2616.